This invention relates to the application of recombinant DNA technology to develop an expression system capable of expressing desired proteins within about one day to about two weeks of transfection. Furthermore, the invention relates to the transformation of a host cell with an expression vector capable of generating stable cytoplasmic mRNA to express a desired protein and vectors capable of expressing trans-activating factors and/or certain translational control effectors, so as to give rise to transient production of the desired protein. The invention further relates to the transfection of selected eukaryotic cells with such vectors such that transient production of the desired protein is obtained.
Recombinant technology has recently been applied to eukaryotic cells, specifically mammalian cells were transformed with heterologous DNA coding for a selectable phenotype. Wigler, M., et al., Cell 11: 223-232 (1977). It has also been shown that eukaryotic cells can be transformed to yield transformants having heterologous DNA integrated into the chromosomal DNA of the eukaryotic cell nucleus.
Successful transformation of eukaryotic cell cultures and expression of DNA sequences coding for a desired protein has been disclosed. See for example, European Patent Publications Nos. 73,659 and 73,656. These successful transformations have utilized vectors to express complimentary DNA (cDNA's) requiring only 5' control signals such as enhancers (Gluzman, Y and Shenk, T. [eds ] Enhancers and Eukaryotic Gene Expression [Cold Spring Harbor Laboratory, 1983]), promoters (Hamer, D. H. et al., Cell 21, 697 [1980]) and 3' polyadenylation sites (Proudfoot, N.J. and Brownlee, G. G., Nature 263, 211 [1976]).
In 1977 it was found that in eukaryotes the cytoplasmic mRNA is not always co-linear with the DNA. DNA sequences encoding proteins were found to be interrupted by stretches of non-coding DNA. There are long stretches of base sequence in the DNA of the gene which do not appear in the final mRNA. It was observed that the primary mRNA transcripts were "spliced" to remove the non-coding sequences, i.e. sequences which do not encode a protein. These non-coding sequences in DNA are generally referred to as introns (formerly referred to as intervening sequences) while the coding sequences are known as exons. RNA polymerase makes a primary transcript of the entire DNA, both exons and introns. This transcript was processed so that the introns were removed while at the same time the exons were all joined together in the correct order. The mechanism producing the foregoing result is referred to as "splicing."
Numerous split or spliced genes have been discovered. In fact, introns exist in virtually all mammalian and vertebrate genes and also in the genes of eukaryotic microorganisms. Introns are not limited to the coding region of a message. For example, one intron was found in the leader region of the plasminogen activator mRNA before the coding sequence in addition to multiple splice sites elsewhere in the gene. Fisher, R. et al., J. Biol. Chem. 260, 1122 (1985). There has been considerable speculation about why introns have evolved and become such a general feature of eukaryotic genes. Crick, F., Science 204, 264, 1979; and, Sharp, P. A., Cell 23, 643-646 (1981).
Given the ubiquity of introns, it is not surprising that splicing was studied in the context of recombinant technology. For example, an SV40 vector was constructed containing a rabbit .beta.-globin cDNA, regions implicated in transcription initiation and termination, splice sites from a multipartite leader sequence located 5' to the .beta.-globin cDNA sequence and a polyadenylation sequence. Mulligan, R. C. et al., Nature 277, 108-114 (1979). This recombinant genome, when infected into monkey kidney cells, was found to produce hybrid mRNAs containing the leader region for the 16S and 19S late RNA and the .beta.-globin coding sequence. This hybrid mRNA produced substantial quantities of the rabbit .beta.-globin polypeptide. Mulligan et al. discuss an experiment in which mutants lacking splicing capability failed to produce discrete mRNAs. Id. at 109.
In an attempt to establish the physiological role that RNA splicing plays in gene expression, Hamer, D. H. and Leder, P., Cell 18, 1299-1302 (1979) manipulated the location and/or presence of a splice site in SV40 recombinants transfected into monkey cells. Hamer and Leder, supra, used one splice site located within the gene encoding the desired protein or used two splice site sequences, one located 5' to and the second within the gene encoding the desired protein. They found that RNA were produced transiently by all of the viruses that retain at least one functional splice junction. They concluded that splicing is a prerequisite for stable RNA formation. Confirming that result, Gruss, P. et al. PNAS (USA), 76 4317-4321 (1979) found that construction of an SV40 mutant lacking an intervening sequence made no detectable capsid protein. These two papers suggest that RNA splicing may be important in a recombinant milieu. However, other studies abandoned splicing to express proteins using only 5' control signals such as enhancers, and promoters and 3' polyadenylation sites. In fact, recent work by Reddy, U. B. et al., Transcriptional Control Mechanisms, J. Cell. Biochem. Suppl. 10D, 154 (1986), found that the inclusion of introns in an expression vector actually reduced the amount of the desired protein expressed.
Straightforward expression using standard recombinant control signals such as enhancers, promoters and 3' polyadenylation sites cannot always be achieved. The SV40 promoter without a splice site has been used to direct expression of numerous cDNAs. (.beta.-galactosidase, Hall, C. V. et al. J. Mol. Applied Genetics 2,; human interferon, Gray, P. W., et al., Nature 295, 503 (1982); hemagglutinin, Gething, et al. Nature 293, 620 (1981); human lecithin-cholesterol acyltransferase, McLean, J. et al., PNAS 33, 2335 (1986); DHFR, Simonsen, C. C. et al., PNAS 80, 2495 (1983); human interleukin-2, Leonard, W. T. et al., Nature 311, 626 (1984); ras-2, Capon, D. J. et al. Nature 304, 1983; src, Snyder, M. A. et al., Cell 32, 891 (1983); and hepatitis B surface antigen, Crowley, C. W. et al., Mol. Cell Biol. 3, 44-55 (1983)).
Transient expression systems have been used as tools of recombinant technology. For example, the analysis of promoter sequences, effects of enhancers, and demonstration of transcription regulation have been facilitated using transient expression systems. One well characterized transient expression system is that for chloramphenicol acetyl transferase (CAT) (Gorman, C. M. et al., Mol. Cell. Biol. 2:1044-1051 [1982]).
Various viral proteins produced in cells infected by DNA viruses are known to activate viral genes expressed during later phases of the temporally regulated lytic life cycle. (Keller, J. M. et al., Cell 36:381-389 [1984]). These proteins include simian virus 40 (SV40) T antigen, adenovirus Ela and Elb protein, the herpesvirus immediate early (IE) proteins and human and simian immunodeficiency viruses. (Benoist, C. and Chamber, P., Nature (Lond.) 290:304-310 [1981]; Hearing, P. and Shenk, T., Cell 39:653-662 [1983]; Rosen, C. et al., Nature 319, 555-559 [1986]). These proteins are the products of genes containing efficient promoters activated by cis-acting elements. Each protein may also have a trans-activating function by activating the expression of other viral genes to permit the virus to progress through its lytic cycle. A transcriptional activation function by increasing expression of other viral genes of each of these proteins has been demonstrated in its respective viral system. Since this transcriptional activation can be provided by cotransfection of a separate plasmid, this effect is referred to as "trans-activation." (Berk, A. J. et al. Cell 17:935-944 [1979]; Brady, J. et al. PNAS [USA] 81:2040-2044 [1984]; Dixon, R.A.F. and Shaffer, P. A., J. Virol. 36:189-203 [1980]; Jones, N. and Shenk. T., PNAS [USA] 76:3665-3669 [1979]). Some data using transient expression with Ela and the IE proteins indicate that these proteins may also trans-activate promoters that are not homologous to their respective viral system. (Green, M. R. et al., Cell 35:137-148 [1983]; Imperiale, M. R. et al., Cell 35:127-136 [1983]). Other data suggests that Ela suppresses some enhancers. (Borelli, E. R. et al., Nature [Lond.] 312:608-612 [1984]).
It is an object of the present invention to provide a transient expression system capable of producing a desired protein. Another object of this invention is to eliminate the time necessary to establish continuous production to obtain a desired protein. It is an object of this invention to provide useful amounts of a desired recombinant protein in about one day to two weeks after transfection. Yet another object of this invention is to provide expression vectors useful in a transient expression system. Still another object of this invention is to provide a host cell capable of being used in a transient expression system to produce a desired protein in about one day to fourteen days of transfection. Another object is to provide certain trans-activating factors and/or translational control effectors capable of enhancing the yields of a desired protein in a transient expression system by stabilizing the transfected DNA.